genes of the extinct caucasian bison still roam the białowieża forest and are the source of...

Genes of the extinct Caucasian bison still roam theBiałowieza Forest and are the source of geneticdiscrepances between Polish and Belarusianpopulations of the European bison, Bison bonasus

MAŁGORZATA TOKARSKA1*, ALEKSEI N. BUNEVICH2, DITTE DEMONTIS3,TARAS SIPKO4, KAJETAN PERZANOWSKI5,6, GENNADY BARYSHNIKOV7,RAFAŁ KOWALCZYK1, YULIYA VOITUKHOVSKAYA8, JAN MAREK WÓJCIK1,BARBARA MARCZUK1, IWONA RUCZYNSKA1 and CINO PERTOLDI9,10

1Mammal Research Institute PAS, Waszkiewicza 1, 17-230 Białowieza, Poland2State National Park, Belovezhskaya Pushcha, Kamieniuki, Białorus3Department of Biomedicine, Aarhus University, Wilhelm Meyers Allé 4, 8000Århus C, Denmark4A.N. Severtsov Institute of Ecology and Evolution RAS, Leninsky pr. 33, Moscow, 119071, Russia5Museum and Institute of Zoology, Polish Academy of Sciences, Wilcza 64, 00-679 Warszawa, Poland6Applied Ecology, Catholic University of Lublin, Konstantynów 1H/402, 20-708 Lublin, Poland7Zoological Museum of the Zoological Institute RAS, Universitetskaya Naberezhnaya 1, St.Petersburg, Russia8Institute of Genetics and Cytology at National Academy of Sciences of Belarus, Akademicheskaya st.,27; 220072 Minsk, Republic of Belarus9Department 18/Section of Environmental Engineering, Aalborg University, Aalborg, Denmark10Aalborg Zoo, Aalborg, Denmark

Received 11 August 2014; revised 14 November 2014; accepted for publication 14 November 2014

European bison (Bison bonasus) populations from both the Polish (PL) and the Belarusian (BY) sides of theBiałowieza Forest represent the Lowland genetic line (LB line) – progeny of the Lowland bison (Bison bonasusbonasus) that inhabited western, central, and south-eastern Europe in historical times. During the speciesrecovery, one of the founders was a descendant of the extinct Caucasian bison (Bison bonasus caucasicus) and itsdescendants formed the other genetic line – Lowland–Caucasian (LC). There have been justified suspicions that LBEuropean bison in the former Soviet Union had undergone cross-mating with the LC line. We performed acomparative genetic analyses on European bison from the BY and PL parts of the Białowieza Forest, the LC lineand extinct Caucasian bison, based on a set of 19 microsatellite markers and 1512 bovine single nucleotidepolymorphism (SNP) markers, polymorphic in at least one of the studied populations. Although genetic variability(mean allele number and expected heterozygosity) for both populations were similar, the FST jack-knifing andprincipal component analyses PCA revealed highly significant differences between PL and BY bison from theBiałowieza Forest. Examining DNA of the extinct Caucasian bison revealed that at least part of the geneticvariants found in the BY, but not the PL, population were of Caucasian origin. The results indicate that thecontemporary population of European bison from the BY part of the Białowieza Forest should not be regarded asa LB line. The results also suggest that the actual global population size of the LB line European bison is only ahalf of its official status. Consideration of the presented results are crucial in determining management actions andpolicy decisions in order to conserve LB line bison within the Białowieza Forest – its natural refuge.© 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ••, ••–••.

ADDITIONAL KEYWORDS: introgression – microsatellites – SNPs.

*Corresponding author. E-mail: [email protected]

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Biological Journal of the Linnean Society, 2015, ••, ••–••. With 3 figures

© 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ••, ••–•• 1

mailto:[email protected]

INTRODUCTION

The decision to recover the Lowland bison (Bisonbonasus bonasus L.) was made shortly after its extinc-tion in the wild in 1919 and took place in Białowieza,Poland (Krasinska & Krasinski, 2013). As well as theLowland bison individuals, a descendant of one of thelast representatives of the Caucasian bison (Bisonbonasus caucasicus) (M 100 KAUKASUS) was used inthe recovery process (Pucek et al., 2004). This decisionserved to save some of the Caucasian bison genesfrom complete loss. All the restored individuals of theEuropean bison have been divided into two geneticlines: the Lowland line (LB line) with animals of pureLowland origin and the Lowland-Caucasian line (LCline) with individuals of mixed LC origin. The extantLB line has seven Lowland founders (Slatis, 1960),while the LC line originates from 11 Lowland bisonand one Caucasian bull (Pucek et al., 2004). Keepingthe lines separate has been a priority of the Europeanbison conservation strategy (Pucek et al., 2004),established to save the higher genetic potential of theLC line (Pucek et al., 2004). The higher genetic poten-tial of the LC line is not necessarily reflected by itshigher fitness. In fact, in spite of its almost half-lowerinbreeding level (approx. 26% versus almost 50% inLB line) there are reports of inbreeding depressionsymptoms affecting reproduction and health param-eters of LC line individuals (Olech, 2003). The rela-tively higher fitness of the LB line appears to be aneffect of the randomly selected high quality genomesof the seven founders. The presumed lowered fitnessof the LC line may be explained as a result of disad-vantageous genetic variants inherited from one ormore of the Lowland founders, or may be an effect ofoutbreeding depression between Lowland and Cauca-sian founders.

Up to now, the demographic status of the LB line ofEuropean bison has seemed satisfactory. According tolast European Bison Pedigree Book (Raczynski, 2012)there are almost 5000 European bison worldwide andmore than half of them belong to the LB genetic line.The vast majority of them (83%) lives in seven free-roaming herds, considered ‘demographically safe’populations (over 100 individuals each). Aside fromtwo free-roaming LB populations in the Polish (PL)and Belarusian (BY) parts of the Białowieza Forest,there are three more in Poland (Puszcza Borecka,Puszcza Knyszynska, Stado Zachodniopomorskie) andtwo more in Belarus (Osipovichskij Opytnyjj Leskhozand Ozery Grodnenskogo Rajjona). Another threefree-ranging LB herds are about to reach the thresh-old of 100 individuals: Landshaftnyj ZakaznikNalibokskij and Polesskij Radiacionno-EkologicheskiZapovednik in Belarus as well as Panevežys-PašiliuStumbrynas in Lithuania (Raczynski, 2012). Accord-

ing to Krasinska & Krasinski (2013) all the aforemen-tioned BY and Lithuanian herds were founded usingbison originating from the Prioksko TerassnyjReserve (Russia) and BY herd from Białowieza Forest(Belarusian Belovezhskaja Pushha) (Bunevich,Krasinska & Daleszczyk, 2006; Krasinska &Krasinski, 2007). According to EBPB data on the endof 2012, there were 23 free-ranging herds of Europeanbison belonging to LC line including: 1 Polish, 12Russian, 1 Slovak, and eight Ukrainian herds. Eightsemi-free herds of LC line were at: Bulgaria (1),Germany (1), Latvia (2), Romania (2), Russia (1) andUkraine (1).

European bison from the PL and BY parts of theBiałowieza Forest form the two largest populations ofthe LB line in the world (Raczynski, 2012). They arepermanently isolated by a border fence (2.5 m highbarbed wire fence) established in 1981 for politicalreasons (Fig. 1) (Bunevich et al., 2006; Kowalczyk,Schmidt & Jedrzejewski, 2012). The contemporary BYbison population in Białowieza Forest descenddirectly or indirectly (via former Soviet Union herds)from 46 individuals restored in Poland (Bunevich,2004; Bunevich et al., 2006; Krasinska & Krasinski,2007). Twenty three of them came directly fromBiałowieza (Poland), two from Pszczyna (Poland) and18 from Pryoksko-Terrasnyj breeding centre (formerSoviet Union) (Bunevich et al., 2006).

In contrast with Poland, bison from both geneticlines were not consistently separated in the formerSoviet and present-day BY part of Białowieza Forestand Prioksko-Terrasnyj Zapovednik. Although thebison herd managers at that time tried to preventanimals of different genetic lines from potentialintrogression, it have remained unknown whetherthe BY population from the Białowieza Forest hasLC genes admixture or not (Bunevich et al., 2006;Pucek, personal communication). European bisonfrom the present BY part of Bialowieza Forest aswell as other BY and Lithuanian populations areconsidered representatives of the LB genetic line.Based on the official genealogy and genetic variationsimulations, Daleszczyk & Bunevich (2009) sug-gested that PL and BY herds from Białowieza Forestshould be allowed to merge to increase the geneticpotential of the whole Białowieza Forest populationof European bison.

There have been several reports of a single bisoncrossing the border prior to 1981. The first observedborder crossing was by two males in 1963 andwas recorded by Korochkina (1969). In the 1970s,Bunevich reported 155 individuals entered and 112left the BY part of Białowieza Forest (Bunevich,2003). There were also several reports of singleindividuals crossing the PL-BY border (Z. Krasinski,personal communication). However, no comparative

2 M. TOKARSKA ET AL.

© 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ••, ••–••

genetic study has been performed on the populationsof European bison from the Białowieza Forest tosupport or deny the potential introgression.

The genetic status of the population of Europeanbison from the BY part of the Białowieza Forest hasnever been officially questioned although doubts werenot isolated and rather well founded, based on theunclear history of European bison recovery in theformer Soviet Union. The possibility of crossbreedingbetween LB and LC lines was also shown by resultsof earlier genealogical (Sipko, 2002), immunogenetic(Sipko et al., 1993, 1995) and morphological (Rautian,Puzachenko & Bunevich, 2003) studies. The ultimateconfirmation was to be delivered by genetic studies(Bunevich et al., 2006).

We undertook this study to verify the existing butunproven reports of crossbreeding between the twogenetic lines of European bison in the former SovietUnion herds and to clarify the genetic status of theEuropean bison from the BY part of the BiałowiezaForest. To achieve that, our succeeding aims were: (a)to describe genetic composition of the PL and BY

European bison from the Białowieza Forest; and (b) tocompare the genetic composition of them with thegenetic composition of LC line and of the extinctCaucasian bison.

MATERIAL AND METHODSTISSUE SAMPLING AND DNA EXTRACTION

Tissue samples were collected from European bison in-dividuals living in the PL (N = 247) and BY (N = 48)parts of Białowieza Forest between 1990–2012. Thesamples were collected from either museum speci-mens, during annual culling or immobilization forvery high frequency and GPS-tracking. Hair samplesof individuals of the LC bison line living in theBieszczady Mountains (Southern Poland) (N = 26) andtissue samples of LC line individuals from Russianreserves (N = 30) were collected. As a reference, skullbones of (N = 10) Caucasian bison Bison bonasuscaucasicus (N = 10) from a collection of the ZoologicalMuseum of the Zoological Institute RAS in St.

Figure 1. Outline of the Białowieza Forest. The border between Poland and Belarus that goes along the border withinthe Białowieza Forest range divides the world’s biggest European bison population into two. The fence practicallyprevents migrations of wild ungulates on both sides and has done for over 30 years.

CAUCASIAN GENES IN THE BIAŁOWIEZA FOREST 3


Petersburg (Russia) were sampled and analyzed.Those skulls were obtained during the late 19th andearly 20th century Tzars’ hunts and stored as trophies.

The DNA of the Caucasian bison was extractedfrom horn bone scratches and fragments of spongybones of the nasal passages, using a Sherlock kit(A&A Biotechnology, Gdynia, Poland) or 6.6% Chelex(Bio-Rad). The DNA of the contemporary Europeanbison was isolated using either the DNeasy Blood orTissue Kit (Qiagen, Venlo, The Netherlands, Cat. no.69506), Sherlock AX (A&A Biotechnology, Gdynia,Poland), or Chelex 100 (Bio-Rad, Hercules, CA, USA,Cat. no. 142–1253) protocols. The DNA concentrationwas subsequently measured with a NanoDrop 1000Spectrophotometer (Thermo Scientific).

The extracted DNA was analyzed in the MRI PASlaboratory (microsatellite loci) or sent for bovinesingle nucleotide polymorphism (SNPs) microchipgenotyping (http://arosab.com).

GENOTYPING

Microsatellite markersNineteen cattle markers used previously in Americanand European bison studies (Wilson, Olson &Strobeck, 2002; Tokarska et al., 2009a, b) (Table 1)were genotyped in all collected samples. Genotypingof the BY bison was carried out following the proce-dures described in Tokarska et al. (2009a, b). Poly-merase chain reactions (PCR) for the LC bison (forhair bulb DNA) and the Caucasian bison (horn bonescratches and spongy bones of the nasal passage)were carried out in total volume of 6 μl, consisting of2.5 μl Hot Start Master Mix Polymerase (Qiagen),20–50 ng of DNA, 1 pmol of each primer anddeionized water to make a final volume of 6 μl.Amplification of the PCR product was carried out infollowing conditions: 95 °C for 15 min, then threecycles of: 94 °C for 2 min, 54 °C for 20 s and 72 °C for10 s, followed by 36 cycles of: 94 °C for 15 min, 54 °Cfor 20 s, 72 °C for 10 s and a final elongation step at72 °C for 30 min.

SNP markersSNP genotypes were obtained for a panel of 50 bisonfrom the PL part of the Białowieza Forest (describedin Tokarska et al., 2009b), nine individuals from theBY part of the Białowieza Forest and eight LC linebison from the Bieszczady Mountains (Poland). Pro-cessing and genotyping of the SNPs were performedusing BovineSNP50 BeadChip (Illumina), whichallows genotyping of SNPs across the entire bovinegenome. There were 50 000 SNPs, before frequencyand genotyping pruning. Genotypes were called usingGenomeStudio software, according to the manufactur-er’s protocol (Infinium II Multi-Sample), as described

in Pertoldi et al. (2009). All the bison SNPs werecalled using the bovine cluster separation file. Unre-liable samples were rejected after analysis of samplecall frequency. To ensure correct calling of genotypesall SNPs segregating in the European bison werechecked manually. Only clusters located within thesame range of intensity as cattle were accepted inorder to identify reliable genotypes. Additional strin-gent quality control of the called clusters was per-formed including removal of monomorphic SNPs,SNPs with low intensities, SNPs with low clusterseparation scores and SNPs with no call. Afterquality control (QC), 1516 SNPs remained for furtheranalyses.

DATA ANALYSIS

Microsatellite markersAllele frequencies and expected heterozygosity (HE)were estimated using GenAlEx vers. 5.5 software(Peakall & Smouse, 2012). Deviations from theHardy–Weinberg equilibrium (HWE) were estimatedusing Genepop (Rousset, 2008) and GenAlEx (Peakall& Smouse, 2012). Genetic divergence (FST) values andbootstrapped confidence intervals were estimatedusing FSTAT (Goudet 2001). Due to high inbreedingof the European bison population we decided to inves-tigate population structure using PCA methods ofGenAlEx, which do not assume Hardy–Weinberg andlinkage equilibria.

We estimated effective population size (NE) valueswith ONeSAMP software based on Bayesian compu-tation (Tallmon et al., 2008). The significance of dif-ferentiation between and among pops was testedusing analysis of variance (ANOVA) (XLSTAT forExcel, version 2014.4.10, Addinsoft, Paris, France).

SNPsOf the 53 851 cattle SNP loci on the BovineSNP50BeadChip that amplified in the European bison, 1516loci were polymorphic in at least one of the threeanalyzed populations. We found deviations from HWEin nearly 9% of the studied loci (in the PL and BYpopulations). A genotype call rate > 95% was appliedto all SNPs and the final combined breed dataset wasof 1516 polymorphic SNPs.

This dataset was used to estimate the geneticvariation, including HE, calculated across all loci(within a population) and percent polymorphic loci(P%) using GenAlEx software (Peakall & Smouse,2012).

We evaluated eventual presence of geneticsubstructuring of all the data sets (PL, BY and LCpooled) with PCA using the GenAlEx software. Inaddition, genetic divergence (FST) was estimatedbetween the three populations.



http://arosab.com

Tab

le1.

Com

pari

son

ofn

um

ber

ofal

lele

s(N

a)an

dn

um

ber

ofef

fect

ive

alle

les

(Ne)

betw

een

Pol

ish

(PL

)an

dB

elar

usi

an(B

Y)

Eu

rope

anbi

son

popu

lati

ons

from

the

Bia

łow

ieza

For

est

and

Low

lan

d–C

auca

sian

(LC

)bi

son

lin

e.N

stan

dsfo

rsa

mpl

esi

ze;H

ofo

rob

serv

edh

eter

ozyg

osit

y;H

Efo

rex

pect

edh

eter

ozyg

osit

y;an

dH

WE

for

Har

dy–W

ein

berg

equ

ilib

riu

m(h

eter

ozyg

ote

defi

cien

cy).

‘Mea

nfo

rL

C*’

con

tain

sva

lues

afte

rsu

btra

ctin

gth

eei

ght

loci

wit

hex

cess

ive

mis

sin

gda

tain

the

LC

popu

lati

ons

(BM

C12

22,

RT

29,

BB

J24,

BO

VF

SH

,B

M18

24,

AG

LA

269,

BM

1225

and

TG

LA

261)

Loc

us

PL

BY

LC

NN

aN

eH

oH

EH

WE

NN

aN

eH

oH

EH

WE

NN

aN

eH

oH

EH

WE

TG

LA

5724

53

1.20

60.

171

0.17

1n

s48

31.

753

0.33

30.

434

ns

564

1.57

50.

368

0.36

8n

sB

M72

324

33

1.41

10.

267

0.29

2**

482

1.15

60.

146

0.13

7n

s53

21.

636

0.45

30.

392

ns

BM

3507

242

11

0.00

00.

000

M48

11

0.00

00.

000

M53

21.

120

0.11

30.

108

ns

ET

H15

224

55

1.92

70.

408

0.48

2**

*48

41.

564

0.39

60.

364

ns

513

1.50

20.

392

0.33

8n

sB

M28

3024

34

2.57

30.

588

0.61

3n

s48

32.

907

0.81

30.

663

ns

483

2.18

20.

479

0.54

7n

sB

MC

1222

225

31.

143

0.09

80.

125

**48

11

0.00

00.

000

M42

31.

101

0.04

80.

093

ns

BM

5004

247

21.

106

0.10

10.

096

ns

484

1.16

10.

104

0.14

0n

s56

51.

889

0.44

60.

475

ns

RT

2924

52

1.19

50.

163

0.16

4n

s48

21.

280

0.25

00.

221

ns

302

1.34

20.

233

0.25

9n

sT

GL

A53

244

31.

915

0.38

90.

479

**48

31.

947

0.54

20.

491

ns

543

2.03

70.

426

0.51

4n

sB

M75

724

34

2.03

30.

564

0.50

9**

484

2.10

90.

500

0.53

1n

s53

52.

794

0.52

80.

648

ns

BB

J24

244

21.

033

0.03

30.

032

ns

483

1.06

50.

063

0.06

2n

s30

21.

220

0.20

00.

183

ns

BO

VF

SH

198

32.

022

0.38

40.

507

*48

21.

997

0.50

00.

504

ns

262

1.99

70.

423

0.50

9n

sT

GL

A12

624

33

1.84

50.

457

0.45

9n

s48

31.

474

0.27

10.

325

ns

566

2.87

10.

456

0.65

8**

AG

LA

232

244

21.

008

0.00

80.

008

M48

21.

021

0.02

10.

021

M55

11

0.00

00.

000

MB

M44

4024

12

1.20

40.

187

0.17

0n

s48

21.

133

0.12

50.

118

ns

556

1.92

70.

509

0.48

5*

BM

1824

242

31.

592

0.37

20.

373

ns

482

1.75

30.

500

0.43

4n

s30

42.

293

0.63

30.

573

ns

AG

LA

269

243

11

0.00

00.

000

M48

11

0.00

00.

000

M28

31.

500

0.32

10.

340

ns

BM

1225

239

32.

893

0.58

60.

656

ns

483

2.26

80.

688

0.56

5n

s30

32.

341

0.40

00.

582

ns

TG

LA

261

228

41.

202

0.14

90.

168

*48

21.

133

0.12

50.

118

ns

293

1.59

10.

379

0.37

8n

sM

ean

2.79

1.54

30.

259

0.27

92.

471.

512

0.28

30.

273.

260

1.78

50.

358

0.39

2M

ean

for

LC

*3.

636

1.86

60.

379

0.41

2



The number, the mean frequency expressed inpercent (%), and the upper and lower limit of the 95%confidence interval of the private alleles were esti-mated for the PL, the BY and the LC populations.These analyses were repeated by pooling two popula-tions at time in all possible combinations (PL + BY),(PL + LC) and (BY + LC).

For both marker types (microsatellites and SNPs)we performed genetic structure analyses usingGeneClass2 (Piry et al., 2004) and Structure(Pritchard, Stephens & Donnelly, 2000), to testassignment and detect potential migrants.

RESULTSMICROSATELLITE MARKERS

Only the PL population deviated significantly fromHardy-Weinberg Equilibrium at one locus (ETH152)after multiple testing Bonferroni correction (P < 005)(Rice, 1989). Percentage of deviating loci was esti-mated to be 36.8% for PL and 10.5% for LC. Thevalues of NE estimated for the PL and BY populationof the European bison reached respectively 21.8 (95%CI = 16.5–21.4, N = 71) and 32.7 (95% CI = 24.9–45.6,N = 24). The jackknifed FST for the two populationswas estimated at 0.016 (99% CI: 0.002–0.039) indi-cating significant genetic differentiation betweenthem. The significance of differentiation amongstudied populations was confirmed by ANOVA(XLSTAT for Excel). P values were lower than 0.0001for all the pops configurations, either pairwise oroverall.

Out of 19 analyzed microsatellites and three Euro-pean bison populations, the highest percentage ofpolymorphic loci was found in the LC line (94.74%),followed by the PL (89.47%) and BY (84.21%) popu-lations from Białowieza Forest. In addition, meanallele number and mean HE (in brackets) werehighest for the LC line: 3.26 (0.39), followed by the PL[2.79 (0.28)] and BY [2.47 (0.27)] populations, respec-tively (Table 1). The lowest pairwise FST (0.016; 99%CI: 0.002–0.039; standard error (SE): 0.007) was esti-mated for PL and BY bison, then for PL and LC(0.062; 99% CI: 0.033–0.111; SE: 0.014) while the FST

value for BY and LC was estimated at 0.066 (99% CI:0.032–0.105; SE: 0.015). The differences in allelicfrequencies are shown in Table S1. In five of theexamined loci in the BY population, we found alleleswhich were not observed in the PL population. Threeof these alleles were found in the LC line bison(Table 2). We analyzed the five microsatellite loci inthe Caucasian bison samples to verify the hypothesisthat the alleles found in the BY and LC line popula-tions (but absent in the Lowland line in Poland) areof Caucasian origin. The small sample size for Cau-

casian bison could affect the efficacy of this verifica-tion, but in spite of this we found that two alleles atlocus BM5004 found in the BY and LC populationsexisted in Caucasian bison DNA (Table 2).

The number of private alleles found were lowest forBY (3), followed by PL (10) and LC (18). The meanfrequency of the private alleles expressed in percent(%) ranged from 0.45% (PL); through 0.8% (BY) to3.5% (LC).

The first, second and third component obtainedfrom microsatellite – based PCA explained, respec-tively: 67.8%, 8.61% and 7.4%, of the variance (Fig. 2).The PCA revealed a higher genetic differentiationbetween LC individuals and PL/BY than between thePL and BY populations, which are overlapping. Vari-ation within the BY population lies entirely withinthe PL range while the LC population partly overlapswith either PL or BY individuals (Figs. 2a-c).

SNP MARKERS

No loci in the three populations differed significantlyfrom Hardy-Weinberg Equilibrium after Bonferronicorrection (P < 005). The complete list of HWE testoutputs is for each locus and each population is pre-sented in Table S2. The HE found for the BY popula-tion was the lowest across all the populations(HE = 0.171), followed by the PL population(HE = 0.186) and the LC population (HE = 0.246). TheP% values found for the BY population was the lowest(P% = 51.19) followed by PL (74.01%) and LC(74.08%) (Table 3). Markers IDs and allelic frequen-cies of all the 1516 SNPs used in the study in PL, BYand LC populations are presented in Table S4 ofSupplementary Material section.

The number of private alleles found were, as formicrosatellites, lowest for BY (9), followed by PL (289)and LC (360), (Table 4). The mean frequency of theprivate alleles ranged from 3.3% (PL) to 20.6% (LC),(Table 4). The pooling of (BY + LC) showed a moder-ate number of private alleles (27 alleles) followed by(PL + LC) (91 alleles) and lastly (PL + BY) which had95 private alleles (Table 4).

The variation explained by the first, second andthird eigenvalue obtained from PCA were 15.9%, 4.9%and 4.5%, respectively, and in total the PCA revealeda high differentiation between LC and the two otherpopulations PL and BY which are genetically muchmore similar and partially overlapping. It should benoted that the individuals belonging to the LC popu-lation are much more heterogeneous when comparedto the individuals belonging to the PL and BYpopulation. It is noteworthy that one individual fromthe LC population overlapped with the individuals ofthe BY on each of the three first axes of the principalcomponents analyses (Fig. 3A–C).



Table 2. Comparison of chosen microsatellite loci variability in the Polish (PL) and Belarusian (BY) bison from theBiałowieza Forest, the Lowland–Caucasian (LC) genetic line from Poland and Russia and in the extinct Caucasian bisonfrom the Caucasus Mountains, living at the turn of the 19th and 20th centuries. Microsatellite variants absent in the PLbison population but found in the BY bison from the Białowieza Forest (shaded). If the alleles were scored in the LC line,their frequency value is also shaded. Allelic composition of locus BM5004 shows clearly that the alleles found in the BYpopulation are of bull M 100 KAUKASUS heritage. The number of Caucasian bison individuals examined refers to locusBM5004. The number of Caucasian bison analyzed at other shown loci varied from 3 to 7. Framed are microsatellitevariants found in the PL population but not recorded in the BY. If they were scored in the LC line, their frequency valueis also framed

Marker Allele

PL(N = 275)Freq [%]

BY(N = 48)Freq [%]

LC line(N = 59)Freq [%]

Caucasian bison(N = 14)Freq [%]

1 2 3 4 5 6

ETH152 162 6.9 2.1 10.8 0164 66.9 78.1 80.4 0.2166 0 3.1 0 0168 25.7 16.7 8.8 0.8

BM5004 95 0 0 0 0.0799 0 0 0 0.07

101 5.1 03.1 19.6 0107 0 0 0.9 0109 0 1.0 7.1 0.5113 0 3.1 2.7 0.19115 94.9 92.7 69.6 0.07119 0 0 0 0.07139 0 0 0 0.03

BM757 178 0 0 1.9 0180 0 0 1.9 0182 0 2.1 16.0 0188 0 0 0 0.5190 49.8 53.1 33.0 0192 0 0 0 0.09194 0.2 1.0 0 0.41196 49.4 43.8 47.2 0

BBJ24 260 0 1.0 0 0264 98.4 96.9 90.0 0.18266 1.6 2.1 10.0 0.32268 0 0 0 0.5

AGLA232 150 0 0 0 0.5152 0 1.0 0 0154 99.6 99.0 100 0156 0.4 0 0 0.14160 0 0 0 0.36

BM723 152 0.8 0 0 NA154 16.7 7.3 26.4168 26 17 9

BM2830 133 0.2 0 0 NA139 24.5 33.3 8.3149 52.7 40.6 58.3159 22.6 26.0 33.3

BMC1222 263 93.3 100.0 95.0 NA265 0 0 2.4275 6.4 0 2.6

BOVFSH 305 56.1 52.1 48.1 NA307 42.4 47.9 51.9329 1.5 0 0



The pairwise FST values were lowest between PLand BY (FST = 0.034) followed by PL and LC (FST =0.073) and lastly between BY and LC (FST = 0.096),however the FST comparison was only found to besignificant between PL and BY (P = 0.034).

Assignment tests performed using GeneClass2 (notassuming Hardy-Weinberg Equilibrium) confirm SNPmarkers to be much better identification tool in agenetically low-variable species, as the Europeanbison. Altogether, 100% of individuals of the threestudied populations were assigned with 1516 SNPswhile 72% using 19 microsatellites (Table S3). Appli-cation of GeneClass2 for 19 microsatellite data

allowed to detect 95 individuals (with probabilitybelow 0.05) as potential first-generation migrants(Paetkau et al., 2004). The SNP results indicated sixindividuals as potential first-generation migrants(with probability below 0.05) (results for both testsare presented in Table S4).

The structure analysis (Structure 2.3.4) did notclearly indicate the true K, although the likelihoodvalues had their peaks at K = 2 (−6249.0 formicrosatellites and −55813.3 for SNPs), suggestingtwo as the most probable number of clusters. Theresults of Structure analyses for microsatellites andSNPs are presented in Fig. S1.

DISCUSSION

The mean values of basic genetic parameters esti-mated with microsatellites and SNPs (heterozygosity,allele number) for PL and BY populations seeminvariable (Table 1), except for NE, which is almostdoubled in the BY population from the BiałowiezaForest. Nevertheless, despite relatively large popula-tion sizes in the Białowieza Forest (approximately500 in PL part and 450 in BY part), NE values for bothpopulations are extremely low, indicating stronggenetic drift and substantial loss of alleles in bothpopulations. In spite of these similarities and the factthat the reported and acknowledged history of Euro-pean bison suggests they should not differ (Puceket al., 2004; Daleszczyk & Bunevich, 2009; Krasinska& Krasinski, 2013), differences between the two popu-lations in allelic composition and frequency arepresent, statistically significantly differentiating thepopulations.

The probability that the genetic differencesbetween the two populations are a result of sponta-neous mutations seems to be close to zero, asspontaneous germline mutation rates for point sub-stitutions in mammals are approximately 10−8 per bpper generation (Drake et al., 1998). The attempt totrace possible temporal changes in genetic parametervalues in the European bison over the 50 years sinceits reintroduction (Tokarska et al., 2009a) showed nosignificant differences for most of the studied param-eters in the subsequent temporal samples and thus. itseems improbable that the differences between PLand BY bison are a result of a loss of alleles in the PLpopulation. The pairwise differences in FST valuesbased on microsatellite data are, in all cases, highlysignificant. Nominal differences of the jack-knifed FST

values of the population pairs suggest more similari-ties between PL vs. LC than between BY vs. LC.Nevertheless, standard errors of these estimationsimpede such justifications, suggesting that thegenetic diversification of PL and BY from the LCpopulation is comparable. The reason why the FST

Figure 2. Principal component analysis (GenALex) forthe Polish (PL), Belarusian (BY) and Lowland–Caucasian(LC) populations of the European bison, based on 19microsatellite markers. The first two coordinates explain58.91% of the variance and the first three coordinatesexplain 70.85% of the variance. The test was covariance-standardized, based on a Tri Distance Matrix. Figure 2Ashows the first and second components, Figure 2B the firstand third, and Figure 2C the second and third.



value estimated with SNPs was only significant in thePL vs. BY comparison could be due to the low samplesize of the LC population (N = 8).

Surprisingly, the PCA results or STRUCTUREanalyses for both microsatellite and SNP markers donot either support or deny the hypothesis of a LCintrogression record in the ancestry of the BY popu-lation. The strong evidence supporting the hypothesisof crossbreeding between the Lowland line and theLC line was only the presence of Lowland–Caucasianspecific alleles in the BY population. Nevertheless, asthe LC line had four Lowland founders more than theLB line, we cannot exclude that these variants comefrom these Lowland ancestors, even if their geneshares in the extant population are very low (Puceket al., 2004). The comparative analysis with theextinct Caucasian bison revealed that at least some ofthe alleles found in the BY population were quitecommon for Bison bonasus caucasicus at the turn ofthe 19th and 20th centuries (Table 2). This is evidencefor a crossbreeding between LC and LB lines informer Soviet populations of the European bison butalso proof of the presence of ‘Caucasian’ variants inthe BY population.

It is not clear how many individuals took part inthe crossbreeding and where exactly it took place. Itmight have been one individual or more. The resultsindicate that affiliation of the European bison fromthe BY part of the Białowieza Forest as the Lowlandgenetic line is no longer adequate, although not allthe individuals carry the Caucasian bison genes.

The vast majority of the BY herds of Europeanbison originates from the BY part of BiałowiezaForest, therefore genetic profiling would furtherassess the scale of introgression effects.

It is not clear how European bison managers shouldrespond to the results of this study. According to theStatus Survey and Conservation Action Plan for theEuropean bison (Pucek et al., 2004): ‘Pure Lowlandand LC lines should be separated in the wild, as wellas in enclosed breeding centres, up to the time of theirlikely natural contact. This isolation is of particularimportance for preserving the genetic variabilitywithin LC line.’ The authors of the Action Plan couldnot have predicted the situation in the BiałowiezaForest. The separation of the two Białowieza Forestpopulations is now vital in order to protect the world’sbiggest free-roaming population of the Lowland bison.

Table 3. Mean values and standard errors (SE) over loci for number of alleles (Na); number of effective alleles (Ne);observed heterozygosity (Ho); expected heterozygosity (He); and percentage of polymorphic loci (%P) estimated based on1516 SNP loci for Polish (PL), Belarusian (BY) and Lowland–Caucasian (LC) populations (GenAlEx)

Population N Na Ne Ho HE %P

LC Mean 44.59 1.74 1.31 0.18 0.19 74.01SE 0.06 0.01 0.01 0.01 0.01

BY Mean 8.96 1.51 1.29 0.18 0.17 51.19SE 0.01 0.01 0.01 0.01 0.01

LC Mean 7.00 1.70 1.41 0.26 0.24 69.72SE 0.03 0.01 0.01 0.01 0.01

Total Mean 20.18 1.65 1.34 0.26 0.20 64.97SE 0.26 0.01 0.01 0.003 0.003 7.00

Table 4. Mean private allele frequency for the SNP markers expressed in percent (%) and the lower and upper boundof the 95% confidence intervals of the mean (lower 95% – upper 95%). The frequency of the private alleles has beenestimated for the Białowieza population (PL), the Belarusian population (BY) and the Lowland-Caucasian population(LC). In addition the frequency of the alleles has also been estimated for the alleles which have been found present intwo of the three populations investigated. The populations pooled were: (PL + BY), (BY + LC) and (PL + LC). ‘N’ standsfor a number of private alleles in each population

PL BY LC (PL + BY) (BY + LC) (PL + LC)N = 289 N = 9 N = 360 N = 95 N = 27 N = 91

Mean frequency (%) 3.3 9.5 20.6 PL: 20.6 BY: 21.1 PL: 4.5BY: 18.9 LC: 26.4 LC: 25

Low 95%-upper 95% 2.3–4.1 7–12 19–22 PL: 17–23 BY: 11–29 PL: 6.6–11BY: 16–21 LC: 16–34 LC: 21–29



So far, genetic analyses of the PL population fromBiałowieza Forest do not indicate any presence of the‘LC’ genetic variants: this is further supported bygenetic analyzes and recovery history. It is veryunlikely that migrating or trespassing bulls from theBY part of the forest could have effectively bred in PLpart of the Białowieza Forest.

The PCA revealed unexpected variation withinLC bison populations from two different localities,suggesting necessity to study the level of diversifi-cation of the free-roaming isolated populations of LCline.

The study revealed that the official number of free-roaming and so-called ‘genetically safe’ populations ofthe Lowland line bison is considerably overestimated.Out of seven populations, only four free-roamingpopulations in Poland can be regarded as represent-ing the Lowland line. Therefore, the global population

of free-roaming LB bison has been reduced by half(Raczynski, 2012).

According to the results outlined in this study wepropose few recommendations for further conserva-tion of the Lowland line of the European bison:

• The status of the studied BY population in theEuropean Bison Pedigree Book should be adjusted,based on the results of the genetic analyses.

• Further studies are needed to estimate the scale ofintrogression and to determine the genetic status ofall BY populations of the European bison.

• Genetic monitoring of all the free-roaming popula-tions in Poland adjoining the BY border, should beundertaken.

• The application of the presented results in man-agement actions and political decisions seem essen-tial for preservation of the Lowland genetic line ofthe European bison in Białowieza Forest, itsnatural refuge.

ACKNOWLEDGEMENTS

The project was financed by the National ScienceCentre, Poland, Grant No. N N304 358839. The studyhas been partly supported by the Danish NaturalScience Research Council (grant number: #21-01-0526, #21-03-0125 and 95095995) and the AalborgZoo Conservation Foundation (AZCF) for CinoPertoldi.

The authors wish to thank Małgorzata andZbigniew Krasinski for support and consultations aswell as Marcin Górny and Sylwia Czarnomska fortechnical support. We are also very grateful to AmyEycott and Stephanie V. Prior for linguistic improve-ments of the manuscript.

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SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:

Fig. S1. Estimated population structure obtained using the Bayesian software STRUCTURE 2.3.4 for the threesubpopulations of the European bison. Each individual is shown as a thin vertical line, partitioned into K (k –number of predefined clusters) colours representing the proportion of an individual’s membership in each of the



http://www.unil.ch/izea/softwares/fstat.html

clusters. 1 – PL; 2 – BY; 3 – LC. The results were achieved using: A-19 microsatellite loci, B-1516 bovine SNPmarkers (Illumina).Table S1. List of the 19 marker loci, allele frequencies and estimations of Hardy–Weinberg equilibrium (HWE)deviations (heterozygosity deficiency) for each locus and each analyzed European bison population. Abbrevia-tions: ‘ns’ stands for ‘non-significant’; ‘M’ means ‘monomorphic’; and asterisks reflect the level of significanceof deviation from Hardy–Weinberg equilibrium (*P-value 0.01–0.05; **P-value 0.001–0.01; and***P-value < 0.001).Table S2. Hardy-Weinberg equilibrium tests for 1516 SNP marker loci and three European bison populations:PL, BY, and LC, chi-squared values, degrees of freedom (DF), and estimations of Hardy–Weinberg equilibrium(HWE) deviations (heterozygosity deficiency) are estimated for each locus and each analyzed population.Abbreviations: ‘ns’ stands for ‘non significant’; ‘M’ means ‘monomorphic’; and asterisks reflect the level ofsignificance of deviation from Hardy–Weinberg equilibrium (*P-value 0.01–0.05; **P-value 0.001–0.01 and***P-value < 0.001). The values were not corrected for multiple Bonferroni comparisons.Table S3. Results of assignment tests, based on 19 microsatellites and 1516 SNP markers performed for PL,BY, and LC populations using GeneClass2 software. Bolded numbers are individuals assigned to their originalpopulations, regular numbers indicate misassigned individuals.Table S4. Loci names and allele frequencies of 1516 SNP markers in PL, BY, and LC populations.



genes of the extinct caucasian bison still roam the białowieża forest and are the source of...

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